Influenza virus infection continues to be the most important respiratory disease both in terms of morbidity and mortality. The two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), play important roles in tissue and organ tropism, spread, and pathogenicity of influenza viruses. Our long term goals are to understand the molecular mechanisms underlying the balance between the receptor-binding and receptor-destroying functions of the HA and NA and how changes in this balance affect the pathogenicity of influenza viruses in humans. The antiviral effect of a new NA inhibitor, zanamivir, has been demonstrated in clinical trials. However, there is a major gap in our knowledge of the effects of NA inhibitors on properties of the HA and NA of human influenza viruses when replication occurs in a human host. Previous studies on the effect of NA inhibitors on the properties of influenza viruses utilized in vitro cell culture systems without regard to the specificity of the cellular receptors, neglecting the fact that the efficiency of the HA and NA functions strongly depends on the specificity of these receptors (Siaalpha2,3Gal or Siaalpha2,6Gal). Therefore, in Specific Aim 1 we will identify the mechanism(s) that allow human influenza viruses to replicate in the presence of an NA inhibitor in human respiratory tract epithelium containing Siaalpha2,6Gal-receptors. In specific Aim 2 we will test the hypothesis that a decrease in NA activity promotes the replication of zanamivir-resistant viruses with reduced affinity for HA receptors, while in Specific Aim 3 we will determine the effect of HA and NA mutations on the development of zanamivir resistance in vivo, utilizing a ferret model that possesses receptor specificity similar to that of human respiratory epithelium. Finally, in Specific Aim 4, we will determine how changes in the HA and/or NA of zanamivir-resistant mutants affect the virulence of such viruses in ferrets. The major strength of this application is (i) availability of well-characterized zanamivir-resistant mutants isolated from human respiratory tract and (ii) experimental systems with adequate receptor specificity, primary tissue culture of the human respiratory tract epithelium (in vitro) and a ferret model (in vivo). Overall, the proposed studies will provide fundamental information on the interdependence of the two major surface glycoproteins of influenza viruses, focussing on adaptive mechanisms allowing replication under pressure from an NA inhibitor. Improved understanding of HA-NA interactions will undoubtedly aid in counteracting resistance to antiviral agents that specifically target the NA glycoprotein and in elucidating the impact on virulence of such adaptive strategies in human influenza viruses.